Botulinum neurotoxins (BoNT) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. With the current warfare and terrorist activities a vastly enhanced defense against potential bioterrorist weapons is of high priority. BoNTs are potentially significant bioterrorism agents due to their relative ease of production, extreme potency and duration of paralytic activity. Countermeasures are needed to counteract the pathophysiology of BoNTs. In this context, the development of inhibitors of BoNTs would be a important breakthrough for the control of human botulism, and importantly, future terrorist threats. To date there are no current interventions that can reverse the effects of intoxication after the toxin has reached its target inside the cell. As such the overarching goal of our proposal is to uncover molecules that can act within an intoxicated cell to provide symptomatic relief to BoNT/A. There are seven serologically distinct serotypes of BoNT, however, we will only focused on BoNT/A as it exhibits the most sustained intoxication and therefore represents the greatest threat of any of the BoNTs. Working within this framework we have taken a two-pronged approach to define such molecules. The first is based on small non-peptidic molecules that can inhibit the intracellular agent, which causes botulinum neurotoxicity, a protease. For this initiative we will prepare mechanism-based inhibitors as well as try to enhance the potency of our previous lead compounds that have confirmed anti-botulinum properties within an animal assay. Our second initiative relates to the discovery of molecules that when administered will promote acetylcholine release and ones that will promote both acetylcholine release/acetylcholinesterase inhibition, in other words poly-functional intervention. In general we know relatively little about truly effective ways to counter toxin action at the 11th hour, and thus total reliance on any particular intervention is likely to be less than satisfactory. Furthermore, there is a vast difference in the time course of action of current potential antagonist and the toxin. This poses an enormous challenge in terms of discovery of agents for effective antagonism of BoNT/A poisoning. Against this backdrop our research will embrace the discovery of molecules that will provide both immediate and possible long-term relief from these neuroparalytic effects of BoNT/A.